SummaryThe recessive Arabidopsis thaliana fumonisin B1-resistant (fbr6) mutant was identified by its ability to survive in the presence of a programmed cell death (PCD)-inducing fungal toxin FB1. The fbr6 mutant also displays altered plant architecture in the absence of FB1, most notably elongated petioles and enhanced leaf margin serration. These phenotypes are a result of a T-DNA insertion in the SQUAMOSA promoter binding protein (SBP) domain gene, AtSPL14. AtSPL14 encodes a plant-specific protein with features characteristic of a transcriptional regulator, including a nuclear localization signal sequence, a plant-specific DNA binding domain (the SBP box), and a protein interaction motif (ankyrin repeats). A transiently expressed fusion of the AtSPL14 protein to green fluorescent protein is directed to the plant nucleus. DNA sequences immediately upstream of the translation start site direct expression of the b-glucuronidase reporter gene primarily in the vascular tissues, consistent with the phenotypes of the fbr6 mutant. AtSPL14 activates transcription in yeast, with a transactivation domain residing within the N-terminal region of the protein. Recombinant AtSPL14 protein binds A. thaliana genomic DNA in vitro in the absence of other proteins. These results indicate that FBR6/SPL14 functions as a transcriptional regulator that plays a role not only in sensitivity to FB1, but also in the development of normal plant architecture.

Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC 50 values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.

A cadherin-like gene associated with larval midgut tissues was cloned from western corn rootworm ( Diabrotica virgifera virgifera : Coleoptera), an economically important agricultural pest in North America and Europe and the primary target pest species for corn hybrids expressing Cry3 toxins from Bacillus thuringiensis (Bt). The full-length cDNA (5371 bp in length) encodes an open reading frame for a 1688 amino acid polypeptide. The putative protein has similar architecture to cadherin-like proteins isolated from lepidopteran midguts that have been shown to bind to Cry1 Bt toxins and have been implicated in Bt resistance. The D. v. virgifera cadherin-like gene is expressed primarily in the larval midgut and regulated during development, with high levels of expression observed in all instars and adults but not pupae. The corresponding genomic sequence spans more than 90 kb and is interspersed with 30 large introns. The genomic organization of the cadherin-like gene for this coleopteran species bears strong resemblance to lepidopteran cadherins suggesting a common molecular basis for susceptibility to Cry3 toxins in Coleoptera.